The present invention relates to a thermostable phosphatidylethanolamine N-methyltransferase, DNA encoding the enzyme and a process for producing the enzyme. Enzymes of the present invention may be useful for synthesis of a polar lipid with high optical purity and the like.
Phosphatidylethanolamine (PE) N-methyltransferase is an enzyme which catalyzes the PE N-methylation pathway in phospholipid metabolism, by transferring a methyl group of S-adenosyl-L-methionine to an amino group of PE to synthesize phosphatidylcholine (PC) via two intermediates phosphatidyl N-monomethylethanolamine (PME) and phosphatidyl N, N-dimethylethanolamine (PDE) (see FIG. 2).
This kind of enzyme can be obtained by purification from membrane fractions of bacteria, yeast and rat liver or DNA cloning. Their characteristics have been reported (Arondel et al., J. Biol. Chem., 268: 16002-16008(1993); Kodaki and Yamashita et al., J. Biol. Chem., 262: 15428 (1987); Cui et al., J. Biol. Chem., 268: 16655-16663 (1993)). Two distinct enzymes catalyze the PE N-methylation pathway in the metabolism of the yeast Saccharomyces cerevisiae while only one enzyme catalyzes synthesis of PC from PE in the metabolisms of the bacteria Zymomonas mobilis, Rhodohacter spheroides and rat liver. Thus, phosphatidylethanolamine N-methyltransferase is a very useful enzyme for synthesis of PC with high optical purity.
PC is widely used as a main component of liposomal membrane in the studies of, for example, biomembrane or reconstituted membranes. Moreover, PC is contained in various foods as digestible surfactant, and plays an important role in medical and pharmaceutical fields as a component of microcapsule for drugs, low molecular weight compounds or the like. Although phosphatidylethanolamine N-methyltransferases useful for synthesis of PC have been found in bacteria, yeast and rat, most of enzymes are less thermostable since they are derived from mesophilic organisms so that they are not suitable for synthetic reactions that further require use of organic solvents and the like.
Archaea including the genus Pyrococcus contain as a main lipid an ether-type lipid in which glycerol binds to hydrocarbon chain via ether-bond, not via ester-bond. On the other hand, any ester-type lipids, which are main lipids in bacteria and eukaryotes, have not been found in archaea. Accordingly, it has been suggested that phosphatidylethanolamine N-methyltransferase derived from archaea may be associated with synthesis of choline residue contained in ether-type lipid, and thus the enzyme is expected to be useful for synthesis of archaetidyl choline, caldarchaetidyl choline and the like which are remarked as novel components of liposomal membranes. It can be assumed that discovery of an archaea-derived thermostable phosphatidylethanolamine N-methyltransferase that is active in organic solvent will make it possible to develop a novel process for synthesizing polar lipid with high optical purity by introducing a methyl group into PE, archaetidyl ethanolamine, caldarchaetidyl ethanolamine and the like, which are important as lipid components of model membranes.
However, no phosphatidylethanolamine N-methyltransferase that is active under an extreme environment has been obtained and there has been a strong demand for such enzyme.
An object of the present invention is to provide a thermostable phosphatidylethanolamine N-methyltransferase.
Another object of the present invention is to provide a gene encoding the thermostable phosphatidylethanolamine N-methyltransferase as well as a process for producing the enzyme using the gene.
For the above-described purposes, the present inventors noted hyper-thermophilic archaea which are viable at 90-100xc2x0 C., particularly Pyrococcus horikoshii, and obtained a gene that was assumed to show phosphatidylethanolamine N-methyltransferase activity from their genome sequence. Further, the inventors produced the enzyme from the gene by using E. coli and confirmed that the enzyme was stable at a high temperature (90xc2x0 C. or higher) and had phosphatidylethanolamine N-methyltransferase (hereinafter occasionally referred to as PE N-methyltransferase) activity, thus accomplishing the present invention.
This specification includes all or part of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. 11-89312 (filed Mar. 30, 1999), which is a priority document of the present application.
In summary, the present invention relates to the following (1)-(7):
(1) a thermostable enzyme derived from a hyper-thermophilic archaeon Pyrococcus horikoshii, having phosphatidylethanolamine N-methyltransferase activity and an optimum temperature of 90xc2x0 C. or higher;
(2) a thermostable enzyme derived from a hyper-thermophilic archaeon, wherein the enzyme has phosphatidylethanolamine N-methyltransferase activity and the following properties:
(a) optimum temperature: 90xc2x0 C. or higher;
(b) substrate specificity: when the enzyme acts on phosphatidyl N-monomethylethanolamine as substrate, incorporation of a methyl group is detected in only phosphatidyl N,N-dimethylethanolamine but not in phosphatidylcholine, while when the enzyme acts on phosphatidylethanolamine as substrate, incorporation of a methyl group is detected in only phosphatidyl N-monomethylethanolamine and phosphatidyl N,N-dimethylethanolamine but not in phosphatidylcholine;
(c) optimum pH: about 8-9;
(3) a thermostable enzyme having phosphatidylethanolamine N-methyltransferase activity, selected from the group consisting of:
(a) a protein having the amino acid sequence of SEQ ID NO: 1; or
(b) a protein having deletions, substitutions or additions of one or more amino acids in the amino acid sequence of SEQ ID NO: 1, and having phosphatidylethanolamine N-methyltransferase activity and an optimum temperature of 90xc2x0 C. or higher;
(4) DNA encoding the thermostable enzyme of (1) or (2) above;
(5) DNA encoding the thermostable enzyme of (3);
(6) DNA of (5) having the nucleotide sequence of SEQ ID NO: 2;
(7) a process for producing the thermostable enzyme described in any one of (a)-(c), comprising the steps of constructing an expression vector containing DNA described in (4), (5) or (6), transforming a host cell with the vector, culturing the transformed host cell in medium and collecting thermostable enzyme produced.